Bacterial delivery of pro-apoptotic proteins in advanced hepatocellular carcinoma.

总结 目前，对于不能切除的肝细胞癌（HCC）患者尚无根治性治疗方法。 这些病人的预后很差。根据目前的标准治疗方法， 存活时间最多20个月我们建议开发一种安全有效的肝癌治疗方法， 沙门氏菌将活性caspase-3选择性地递送至肿瘤。激活刽子手卡斯帕酶，最后 细胞凋亡的承诺步骤，绕过癌症获得的机制，以避免细胞死亡。系统性 活性半胱天冬酶的递送是不可行的，因为它会引起严重的副作用， 转化成细胞相比之下，沙门氏菌在肝细胞癌中的积累比例为100，000：1，与健康组织相比 结果，可以将具有高特异性的蛋白质递送到癌细胞，同时具有最小的全身副作用。 本研究的概念证明已经通过证明1）细菌递送系统 可以将蛋白质特异性递送到癌细胞中，以及2）活性casp-3的细菌递送诱导细胞死亡 在癌细胞中，并减少小鼠皮下HCC的肿瘤体积。在这份提案中，我们希望 确定细菌递送的活性casp-3（EBT-002）在小鼠中与 标准治疗，并研究与免疫检查点阻断的组合。目标1：量化 EBT-002对皮下HCC肿瘤中细胞凋亡的激活和对肿瘤体积的影响。 原位肝肿瘤模型。在目标2中，我们将确定EBT-002的最大耐受剂量（MTD）， 在剂量递增研究中研究急性毒性，以及在急性肝病背景下研究肝毒性。我们将 测量该疗法在具有肿瘤的小鼠中的生物分布，以确定脱靶细菌积累。 我们还将确定最大有效剂量。我们假设在高细菌剂量下， 与剂量无关。这将能够减少剂量，同时保持相同的疗效， MTD。在目的3中，我们将确定EBT-002对肿瘤中T细胞浸润和肿瘤增殖的影响。 PD-L1的高表达和低表达。这将与抗PD-L1抗体联合检测，以评价 如果这种组合可以增加两种疗法在对以下药物有反应和无反应的肿瘤中的功效， 检查点抑制剂。 该提案的成功结果是EBT-002作为一种新疗法开发的第一步。 HCC。将在II期SBIR中进一步开发该疗法，以获得必要的数据， IND申请。这项研究将建立一个平台技术，直接将蛋白质输送到细胞中， 在信号通路的关键点，并将在广泛的癌症有效。

Summary There are no curative treatments for patients with unresectable hepatocellular carcinoma (HCC) at this time. The prognosis for these patients is dismal. With current standard of care treatment methods, the median survival time is, at best, 20 months. We propose to develop a safe and effective therapy for HCC that employs Salmonella to deliver active caspase-3 selectively to tumors. Activating executioner caspases, the final committed steps of apoptosis, circumvent the mechanisms that cancers acquire to avoid cell death. Systemic delivery of active caspases is unfeasible because it would induce severe side effects and be poorly taken up into cells. In contrast, Salmonella accumulate in HCC in ratios of 100,000 to 1 compared to healthy tissues and, as a result, can deliver proteins with high specificity to cancer cells with minimal systemic side effects. The proof of concept for this study has been established by demonstrating that 1) a bacterial delivery system can delivery proteins specifically into cancer cells and 2) bacterial delivery of active casp-3 induces cell death in cancer cells and reduces tumor volume in subcutaneous HCC in mice. In this proposal, we want to determine the safety and efficacy of bacterially delivered active casp-3 (EBT-002) in mice compared to standard of care and investigate the combination with immune checkpoint blockade. In Aim 1, we will quantify the effect of EBT-002 on the activation of apoptosis in subcutaneous HCC tumors and on tumor volume in an orthotopic liver tumor model. In Aim 2, we will determine the maximum tolerated dose (MTD) of EBT-002 and study acute toxicity in a dose-escalation study as well as liver toxicity in a cirrhotic liver background. We will measure the biodistribution of this therapy in mice with tumors to determine off-target bacterial accumulation. We will also determine the maximum effective dose. We hypothesize that at high bacterial dosage, efficacy is independent of dose. This would enable a reduction of dose, while maintaining the same efficacy measured at MTD. In Aim 3, we will determine the effect of EBT-002 on T-cell infiltration and tumor proliferation in tumors with high and low PD-L1 expression. This will be tested in combination with anti-PD-L1 antibodies, to evaluate if this combination can increase efficacy of both therapies in tumors that are responsive and non-responsive to checkpoint inhibitors. The successful outcome of this proposal is the first step in the development of EBT-002 as a new therapy for HCC. Further development of this therapy will be pursued in a Phase II SBIR to obtain the necessary data for an IND application. This research will establish a platform technology to directly deliver proteins into cells at critical points in signaling pathways and will be effective in a wide array of cancers.